Core 1: Genetically-Defined Microbe Core

Director: John Kappes, Ph.D. 

Co-Director: Charles O. Elson, M.D.

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Requests from Center investigators will be accepted on a first-come, first-served basis, subject to the requirement that the research be appropriate for the Center project and likely to contribute to its success.

Core 1 Services Request Form

Microbiota Microarray List April, 2012

Relation to Human Microbiome Project genome sequences.

 

Code

% identity

Name

function

Primary Species

Phylum

Class

rIB19

36

UvrD/REP helicase

transc/ transl

Propioni-bacterium acnes

Actinobacteria

Actino- bacteria

14-2

80

isolate 14-2 surface extract – flagellin

motility

Roseburia intestinalis

Firmicutes

Clostridia

3_1_57

100

Flagellin

motility

Lachnospiraceae 3_1_57

Firmicutes

Clostridia

BF30S

30S riboprotein

transc/ transl

Bacteroides fragilis

Bacteroidetes

Bacteroidia

Btheta

87

hypothetical protein

other/ unknown

Bacteroides thetaiotaomicron

Bacteroidetes

CBir1

83

Flagellin 

motility

Butyrivibrio fibriosolvens

Firmicutes

Clostridia

CBir11

46

hypothetical protein/ flagellin

motility

Roseburia inulinivorans

Firmicutes

Clostridia

CBir14

90

GAPDH

metabolism

Lactobacillus salivarius

Firmicutes

Bacilli

CBir19

78

ABC transporter.ATP-binding protein

other/ unknown

Bacteroides vulgatus

Bacteroidetes

Bacteroidia

CBir23

85

elongation factor 1A

transc/ transl

Alistepes shahii

Bacteroidetes

Bacteroidia

Cbir45

59

hypothetical protein/ glycosyl hydrolase

metabolism

Bacteroides eggerthii

Bacteroidetes

Bacteroidia

CBir5

63

methyl-accepting chemotaxis protein

motility

Helicobacter cinaedi

Proteobacteria

Delta/ Epsilon

CBir56

60

methyl-accepting chemotaxis protein

motility

Helicobacter canadensis

Proteobacteria

Delta/ Epsilon

CBir63

47

Ig-like surface protein/ hypothetical protein

other/ unknown

Roseburia intestinalis

Firmicutes

Clostridia

CBir66

66

Flagellin/ hypothetical protein

motility

Roseburia intestinalis

Firmicutes

Clostridia

CBir8

90

elongation factor Tu

transc/ transl

Tannerella sp.

Bacteroidetes

Bacteroidia

CT-B

n/a

cholera toxin B subunit

other/ unknown

Vibrio cholerae

Proteobacteria

Gamma

EF20

100

Sal A

other/ unknown

Enterococcus faecalis

Firmicutes

Bacilli

Fla 2

72

flagellin 2 from A4 isolate

motility

Roseburia intestinalis

Firmicutes

Clostridia

Fla 3

81

flagellin 3 from A4 isolate

motility

Roseburia inulinivorans

Firmicutes

Clostridia

Fla X

56

flagellin

motility

Roseburia inulinivorans

Firmicutes

Clostridia

Flic2

0

flagellin

motility

Candidatus arthromitus (SFB)

Firmicutes

Clostridia

Keto

metabolism

Bacteroides fragilis

Bacteroidetes

Bacteroidia

MDR247

54

adenine deaminase 

metabolism

Clostridium bolteae

Firmicutes

Clostridia

MDR254

73

flagellin protein 

motility

Flavonifractor plautii

Firmicutes

Clostridia

MDR90

66

collagen adhesion protein 

other/ unknown

Roseburia intestinalis

Firmicutes

Clostridia

OmpC

100

OmpC from UNC101 E. coli

other/ unknown

Escherichia coli

Proteobacteria

Gamma

rIB10

48

Nucleotidyltransferase/ hypothetical

transc/ transl

Roseburia intestinalis

Firmicutes

Clostridia

rIB12

60

homoserine dehydrogenase

metabolism

Flavonifractor plautii

Firmicutes

Clostridia

rIB13

98

protease IV /SppA

other/ unknown

Bacteroides vulgatus

Bacteroidetes

Bacteroidia

rIB14

84

helicase

transc/ transl

Ruminococcus sp

Firmicutes

Clostridia

rIB15

51

amidohydrolase

metabolism

Clostridium difficile

Firmicutes

Clostridia

rIB16

74

pyruvate synthase

metabolism

Flavonifractor plautii

Firmicutes

Clostridia

rIB17

39

NlpC/P60

other/ unknown

Flavonifractor plautii

Firmicutes

Clostridia

rIB18

30

surface array protein

other/ unknown

Campylobacter showae

Proteobacteria

Epsilon

rIB2

61

SAM domain protein

transc/ transl

Blautia hansenii

F&B

rIB20

0

ABC transporter

other/ unknown

Verminephrobacter eiseniae

Proteobacteria

Beta

rIB4

63

relaxase

transc/ transl

Clostridium asparagiforme

Firmicutes

Clostridia

rIB5

37

hypothetical protein – cytoplasmic

other/ unknown

Erysipelotrichaceae 3-1-53

F & P, chordata

Mollicutes

rIB6

47

hypothetical protein/ bIF-2

transc/ transl

Clostridium leptup

Firmicutes

Clostridia

rIB7

76

malonyl CoA carrier

metabolism

Bacteroides sp.

Bacteroidetes

rIB8

59

hypothetical protein/ glycosyltransferase

metabolism

Lachnospiraceae 3-1-57

Firmicutes

Clostridia

rIB9

45

methyl-accepting chemotaxis protein

motility

Roseburia intestinalis

Firmicutes

Clostridia

SFliC

n/a

Salomonella dublin Flagellin

motility

Salmonella dublin

Proteobacteria

Gamma

UFliC

100

FliC from UNC101 E. coli

motility

Escherichia coli

Proteobacteria

Gamma

Proteins are resuspended at 0.2 mg/ml in 1mM Tris, 0.1% SDS.  The proteins are then be printed onto FAST 16pad slides (Whatman) using a MicroGrid II robot (Genomic Solutions).    The printed slides are allowed to air-dry over night, then blocked with Protein Array Blocking Buffer (Whatman).  Individual pads on the array are incubated with sera and secondarily with Alexa 647-anti-human IgG and Alexa 546- anti-human IgA.   The resulting fluorescent slides are analyzed using a Genepix 4000B  dual laser microarray reader (Molecular Devices) and Genepix Pro 6.0 software.  

The software determines the net median pixel intensities for each individual feature (antigen spot) from a set of 10 measurements/feature.  A median net digital fluorescence unit (DFU) for each feature represents the median values from 8 replicate antigen features on each array.  A software program called “Significance Analysis of Microarrays” (SAM) (14, 23), which was developed at Stanford University will be applied to the data to identify antigens with statistically significant differences in antibody reactivity between groups.  SAM computes a statistic for each feature, measuring the strength of the relationship between the feature intensity and the group.  The software estimates a false discovery rate (FDR), i.e., a false positive rate, for each antigen by permuting the repeated measurements between groups.  SAM results can be further analyzed using software called Cluster and Cluster results can be displayed using Tree View.

Retroviral Unit (Dr. Kappes)

  1. Molecular construction of gene expression vectors (preparation of DNA;  restriction  analysis of DNA; insertion of gene; confirmation of gene expression; large-scale [Maxi prep] production of DNA.
  2. Validation of vector trans-gene expression (DNA transfection and confirmation of gene expression.
  3. Packaging of the vector and cryopreservation of the virus vector stock  (preparation of DNAs for packaging and Env constructs; transfection of mammalian cells; collection and preparation of culture supernatant [virus vector]; cryostorage).
  4. Concentration of the virus vector stock (large-scale transfection of mammalian cells; collection and preparation of culture supernatant [virus vector]; cryostorage).
  5. Generation and titration of GFP-virions (preparation of DNA; large-scale transfection of mammalian cells; collection and preparation of culture supernatant [virus vector]; cryostorage).
  6. Generation of HIV-1 stocks.
  7. Infectivity titration of HIV-1 stocks.
  8. Production of a stable gene-expressing cell line (packaging of vector DNA; transduction of mammalian cells; selection).
  9. Production of a clonal gene-expressing cell line (packaging of vector DNA; transduction of mammalian cells; selection; limiting dilution cloning; expansion of [~25] clones).
  10. Production of a shRNA-expressing cell line.
  11. Confirmation of gene-expression cell line (analysis of cell line for trans gene expression by FACS or immunofluorescence, etc.).

Bacterial Unit (Dr. Elson)

  1. Analysis of bacterial flora via 16S ribosomal DNA for validation of monocolonization or germ-free status (PCR and agarose gel; PCR and DGGE; Elution of bands, cloning, and sequencing).
  2. Collection, 16s RNA characterization and cryopreservation of  bacteria.
  3. Cloning of genes for bacterial expression (PCR, cloning, screening, sequencing).
  4. Expression and provision of bacterial protein relevant to inflammatory bowel disease, per protein.
  5. Quantitation of microbial groups, for up to 25 samples, by quantitative PCR.
  6. Printing antigen microarrays, per slide.